Biological membranes undergo continuous remodeling in processes that transfer material between organelles while maintaining organellar identity and affect the assembly and reassembly of organelles necessary for progression through mitosis. The main objective of the work in this laboratory is to elucidate the mechanisms that regulate the membrane remodeling involved in these processes. The role of the Arf family of GTP-binding proteins has been our focus. The hypothesis being tested is that regulating hydrolysis of GTP on Arf in turn regulates the consumption of protein coated vesicles that mediate membrane remodeling. Consistent with this model, a GTPase- activating protein (GAP) for Arf has been found to be specifically dependent on two phospholipids, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate. The GAP has been purified and cloned. The sequence has revealed a number of domains that are likely relevant to function and regulation, including a 350 a.a. overlap with the general vesicular transport factor p115, a PH domain, a Zn finger domain, ankyrin repeats, a proline rich region including type 1 and type 2 ligands for SH3 domain binding, an SH3 domain, a potential cell cycle dependent kinase phosphorylation site and several potential MAP kinase phosphorylation sites. To test our hypothesis that modulation of Arf GAP regulates membrane remodeling, we will begin by focusing on the PH domain and potential phosphorylation sites.